The present invention relates to a method for isolating and purifying transferrin and lactoferrin receptor proteins from bacterial pathogens and to vaccines containing purified transferrin and/or lactoferrin receptor proteins.
There are a number of important bacterial pathogens causing disease in humans and in animals for which effective vaccines are either absent or unsatisfactory. A number of these pathogens are relatively host specific with respect to their ability to cause natural infection. Bacteria such as Neisseria meningitidis, Haemophilus influenzae and Neisseria gonorrhoeae continue to be an important cause of endemic and epidemic human diseases such as meningitis, otitis, epiglottitis, gonorrhea and urethritis. Similarly, Pasteurella haemolytica, Haemophilus somnus and Pasteurella multocida are important causative agents of pneumonic pasteurellosis and infectious thromboembolic meningoencephalitis in cattle. In pigs, Actinobacillus (Haemophilus) pleuropneumoniae is an important causative agent of infectious pneumonia.
Haemophilus influenzae and Neisseria meningitidis are the most common cause of bacterial meningitis in young children. Despite available effective antibiotic therapy, significant mortality and morbidity result from meningococcal infection. The fulminant nature of the infection, coupled with the scant characteristic clinical signs present in children under two years of age, may be a major factor contributing to continuing mortality and morbidity.
Vaccines based on the capsular polysaccharide of the Neisseria meningitidis bacterium were developed after a correlation was observed between the presence of anticapsule antibody and resistance to systemic meningococcal infections. These capsular polysaccharide vaccines are effective against infection caused by organisms from the A, C, Y, and W-135 capsular serogroups of meningococci. However, no effective vaccine is available against the most common serogroup B meningococci. There is a poor humoral response to the capsular polysaccharide vaccines in children less than two years of age who are at the highest risk of infection from endemic disease. Further, capsular vaccines do not provide immunological memory and the duration of immunity is relatively short. Although attempts to overcome the poor immunogenicity by chemical modification and conjunction to tetanus toxoid show some promise, the results have to be considered in light of the demonstrated serological cross-reactivity of serogroup B capsule with human fetal and infant neural tissue. In view of this consideration, development of a polyvalent polysaccharide vaccine that provides sufficiently broad coverage for prevention of endemic meningococcal disease seems unlikely.
Neisseria gonorrhoeae causes gonorrhea which is plaguing the world in epidemic proportions. Development of a gonococcal vaccine is of a high priority.
Bovine pneumonic pasteurellosis, a major cause of economic loss to the cattle industry, is primarily due to Pasteurella haemolytica. The experimental studies and field trials with vaccines containing P. haemolytica have been inconsistent in reducing the incidence and severity of the disease. Infectious thromboembolic meningoencephalitis, an important cause of mortality in feedlot cattle, is caused by Haemophilus somnus. There is currently no effective vaccine for the prevention of this disease. Actinobacillus (Haemophilus) pleuropneumonia causes a contagious pneumonia in pigs which constitutes a major problem for the swine industry throughout the world. Vaccination with crude vaccine preparations have not been successful due to limited protection of heterologous serotypes.
Iron acquisition is essential for the growth and survival of bacterial pathogens in the host and for causing infection. Bacterial pathogens in the mammalian host are confronted with an environment in which the level of iron is extremely low. In the extracellular compartment, iron is sequestered by the proteins transferrin and lactoferrin, which predominate in serum and mucosal secretions, respectively. The ability to compete with lactoferrin and transferrin for iron is thought to be essential for the pathogenesis of many bacterial infections. Most bacteria manufacture iron-chelating compounds known as siderophores to facilitate iron acquisition from their environment. However, several pathogenic bacteria, such as Neisseria meninoitidis, Neisseria gonorrhoeae, and Haemophilus influenzae do not produce siderophores, but rather acquire lactoferrin iron and transferrin iron directly for growth in vitro.
Early observations of meningococci and gonococci by B. E. Holbein, I. W. DeVoe and F. P. Sparling and co-workers demonstrated that these bacteria can grow in the presence of transferrin or lactoferrin proteins and can use iron from these proteins as the sole source of iron for growth. It was further established that separation of the proteins from the cells with a dialysis membrane excluded the use of transferrin or lactoferrin iron, indicating that soluble factors removing iron from lactoferrin or transferrin were not involved, thus suggesting that cell contact was necessary. Studies by F. P. Sparling and D. W. Dyer have demonstrated that mutants specifically deficient in iron acquisition from transferrin are deficient in binding.
The mechanism of iron acquisition from transferrin and lactoferrin has not previously been studied in the bacteria Pasteurella haemolytica, Haemophilus somnus, Pasteurella multocida, Actinobacillus (Haemophilus) pleuropneumoniae or Haemophilus suis.
By virtue of their functions, the transferrin and lactoferrin receptor proteins are located on the surface of the bacteria when in the host and are accessible to large proteins. Thus, the receptors would be accessible to antibody-mediated host defenses. The transferrin and lactoferrin protein receptors are essential for obtaining iron for growth and for survival. Thus, the pathogen cannot lose its transferrin and/or lactoferrin receptors to evade immunity provided by vaccine antigens containing such receptor proteins. Any attempt at such a evasive technique would result in an inability to survive in the host.
The nature of the iron uptake process was not previously known, and identification and characterization of the lactoferrin and transferrin receptor proteins have not previously been possible. Nor have the lactoferrin and transferrin receptor proteins previously been isolated and purified. Further, no vaccine containing the receptor proteins has been previously developed.